It is well-known in the field of immunoassays using dried test elements, that free, uncomplexed labels need to be separated and removed from bound or complexed labels, prior to detection. This is done by applying, after the sample is added, a wash liquid to the test element after the complexing reaction has occurred, to cause chromatographic separation of the free labels from those that are bound. Such separation, in theory, leaves a volume in the test element in which the bound labels can be read free of the interference of the uncomplexed ones that are now washed away. The process is exemplified by the following U.S. Pat. Nos. 4,517,288; 4,752,562; 4,774,174 and 4,786,606. It is well understood that the label can be attached to a immunoreactant for competitive assays, or to an antibody for sandwich assays, either process being of use in this invention.
In practice, however, notwithstanding at least 9 years of existence of such patents, such a process does not always work well. The practice taught by said patents has been to apply a static "stream of (eluting) solvent" to the center of the reaction zone. That is, a dispensing tip is positioned above a test element already wetted with patient sample, above the substantial center of the reaction zone, and a stream continuously pours out onto that center of the reaction zone. The tip stays fixed relative to the test element during washing. It turns out that such a continuous flow produces underneath the center thereof, in certain test elements, a portion in which the free labeled immunoreactant do not get washed out.
The causal mechanism for this artifact was not understood prior to this invention. The artifact meant that reading could not be centered on the spot that was washed, without being affected by this artifact. Indeed, an unwashed center of the wash zone drastically reduces the useful read portion, as will be readily understood.
Still another problem with the conventional stream method of washing has been that unacceptable background rates and/or biases have been detected with certain chemistries. The causal mechanisms for these biases following washing, have also been unknown. The problem of background and biases has been particularly troublesome in immunoassays that are rate assays.
Hence, prior to this invention there has been a long-felt need for a wash protocol that effectively removes the unbound labeled immunoreactants from substantially all of the washed volume, including that centered on the wash liquid that impacts the test element. There has been a further need to eliminate the background "noise" and biases that sometimes occur.
(As used herein, "labeled immunoreactant" means any labeled antibody or antigen that is used to detect a target analyte, such as an antigen or antibody, whether the assay be via a competitive assay in which a target antigen, for example, competes with a labeled version of the antigen, or a sandwich assay in which a target antigen, for example, forms a sandwich between a capture antibody and a labeled antibody (the labeled immunoreactant). The label in turn can be any useful detectable direct or indirect indicator, e.g., a fluorophor or an enzyme, respectively.)